Ionotropic glutamate receptors control a wide variety of normal neuronal processes including learning and memory. Activation of these important neurotransmitter receptors is involved in a number of neurodegenerative diseases, notably stroke and epilepsy. In addition, drugs that enhance the activity of glutamate at the AMPA subtype of glutamate receptors (allosteric modulators) have been shown to improve cognition and may have benefits in neurodegenerative diseases such as Alzheimer's disease. The domain of AMPA receptors that binds glutamate (S1S2 domain) can be produced as a soluble protein in bacteria and studied with a variety of high-resolution biophysical methods. The S1S2 domain has been shown to be an excellent model system for the study of drugs that interact with AMPA receptors in that, at least at the agonist binding site, the binding affinity is the same as the membrane-bound, full-length tetrameric protein and the conformational transitions upon binding seem to be similar as well. The protein is monomeric below 6 mM, but allosteric modulators can promote dimerization of the S1S2 domain, while blocking desensitization and slowing the rate of deactivation in the membrane-bound protein. We have determined the crystal structure of one AMPA receptor-binding domain (GluR2 S1S2) with six different allosteric activators and studied some of the complexes using NMR spectroscopy. Considering the binding interactions made in all of the structures, a relatively large binding surface at the dimer interface can be defined. Despite considerable work on the functional effects of allosteric modulators, very little is known about the mechanism of binding, the structural interactions that lead to a functional outcome, and, in some cases, even the stoichiometry of binding. We propose to define the binding interaction in detail using X-ray crystallography, NMR spectroscopy, isothermal titration calorimetry, and analytical ultracentrifugation, in conjunction with whole cell recording. The goal will be to understand how drugs bind to this surface (monomer vs. dimer, what controls the subsite specificity), how to amplify affinity, how binding interactions are related to functional outcomes, and how to provide as much specificity as possible at this site. In addition, we have designed and are synthesizing dimeric forms of allosteric activators that we anticipate should bind with higher affinity and specificity than currently available drugs. We believe that detailed studies of the interaction of these drugs with AMPA receptors described in this application will lead to new clues as to the design of effective drugs for enhancing cognition. PUBLIC HEALTH RELEVANCE: AMPA receptors mediate the majority of fast excitatory synaptic transmission in the central nervous system. Enhancement of the activity of AMPA receptors has been shown to be beneficial in increasing cognition, such that drugs that amplify the effect of glutamate (allosteric activators) may be effective in treatment of Alzheimer's disease and other neurological disorders. This application is targeted toward understanding the binding site for allosteric activators with the goal of defining the mechanism of action and the structural correlates of the functional effects of these drugs.